Notch信号途径对小鼠骨髓EPC和EOC的调控

发布时间：2019-06-25 15:10

【摘要】：EPC是成熟血管内皮细胞的前体细胞，属干细胞群体，和HSC来自同一祖先细胞。在胚胎，EPC参与胚胎发育的血管发生，出生后，与HSC共同存在于骨髓干细胞龛中，也存在于外周血、脐血中，具有缺血区定向归巢并分化为成熟内皮细胞、促进受损内皮修复、血管新生等重要作用，不仅可应用于对于缺血性疾病的治疗，对肿瘤的防治也有重要价值。 EPC最早是从外周血中分离CD34+细胞得到的，其表面标志被认定为CD34\CD133\KDR，，但是随着进一步的研究发现EPCs是一组异质性的细胞群，它是由众多处于不同分化阶段的细胞组成，它的表面标记随时间改变而变化。研究显示，虽然不同的培养方法和细胞来源使EPC的表面标志不完全一致，但是目前体外培养的EPC有两类，一类是梭型、增殖潜力有限、培养不超过8周的early EPC（EEPC），另一类是鹅卵石样的、具有高增殖潜能、可以持续传代培养的外向型生长内皮细胞late EPC (EOC)。研究显示，EEPC和EOC都具有一定的血管形成和组织修复作用，但EEPC和EOC在血管的形成和受损组织修复的过程中具体发挥什么作用，与哪些信号通路和调控机制有关目前尚不清楚。 Notch信号途径从胚胎发育到成年个体的多个系统中发挥重要作用，对不同组织中的细胞命运起着决定性作用。以往的研究提示Notch信号途径对EPC的定植、迁移和归巢有重要作用，我们前期的实验也表明，小鼠肝脏部分切除后，EPC可以迁移至受损的肝脏组织参与肝组织的再生，但对EOC的影响未见报道。为了进一步分析Notch信号途径对内皮干/祖细胞的调控机制，我们研究Notch信号途径对不同分化阶段的EPC的调控作用，由于转录因子RBP-J是Notch受体下游的关键效应分子，可以介导四种Notch受体在核内的转录激活作用，因此是整个Notch信号途径调控的汇集点。我们通过建立RBP-J条件性剔除小鼠，拟表达了Notch在多种组织中的缺失，研究结果显示Notch信号途径在维持血管平衡和组织修复方面有重要的作用。主要研究成果如下： 1．通过体外贴壁扩增培养，从小鼠骨髓细胞中成功培养出EEPC和EOC，表达CD34+/CD133+/VEGFR2+的EEPC数目从最初的0.08%能够增长至50%以上； 2．我们发现，在三维管腔形成实验中，EEPC不能形成管腔样结构，而EOC能够形成管腔样结构并且受到Notch-RBP-J信号通路的调控；阻断Notch-RBP-J信号通路后，EEPC的增殖，迁移，CXCR4的表达均呈下降趋势而EOC的增殖、迁移以及CXCR4的表达呈上升趋势。提示Notch信号通路对EEPC和EOC的调控不相同； 3．通过RBP-J条件性基因剔除小鼠模型，我们进一步观察到，小鼠肝大部切除术后（PHx），通过Notch信号通路的调控EEPC可以迅速募集到受损肝脏，促进肝脏血管的重建、肝细胞的增殖以及肝功能的恢复，而EOC并没有有效促进肝脏的再生和肝细胞的增殖，提示Notch-RBP-J信号通路在EEPC和EOC参与的肝再生进程中的调控作用不同，并且发现EEPC和EOC在肝再生过程中发挥着不同的作用。综上所述，我们通过RBP-J剔除小鼠，观察了Notch-RBP-J信号对于EEPC和EOC参与的肝再生进程的调控作用。我们的结果表明， Notch信号参与调控EEPC和EOC对肝细胞的再生，肝脏的修复，以及肝功能的恢复，并且EEPC和EOC的增殖、分化和迁移受到Notch-RBP-J信号途径的调控；EEPC和EOC在肝再生进程中发挥着不同的作用。这些研究为进一步了解内皮干/祖细胞的功能，深入分析Notch对内皮干/祖细胞的调控机制奠定了基础，
[Abstract]:The EPC is the precursor cell of mature vascular endothelial cells, the stem cell population, and the HSC from the same ancestor cell. In the embryo, the EPC participates in the angiogenesis of the development of the embryo, and is co-existing with the HSC in the bone marrow stem cell niche after birth, and is also present in the peripheral blood and the umbilicus blood, has the important effects of directional homing and differentiation of the ischemic region into the mature endothelial cells, promoting the repair of the damaged endothelium, the angiogenesis and the like, Not only can be applied to the treatment of ischemic diseases, but also has important value for preventing and treating the tumor. EPC was the first to separate CD34 + cells from peripheral blood and its surface markers were identified as CD34CD133KDR, but with further study, EPCs were a heterogeneous group of cells, consisting of numerous cells at different stages of differentiation, whose surface markers varied with time The results show that, although the different methods of culture and the source of the cell make the surface marker of the EPC not exactly the same, there are two types of EPC in vitro, one is the shuttle type, the proliferation potential is limited, the early EPC (EEPC) with no more than 8 weeks is cultured, and the other is a cobblestone, which has high proliferation potential. an extraversion growth endothelial cell (EOC) EPC (EOC) that can be continuously subcultured ). The study shows that both the EEPC and the EOC have certain vascular formation and tissue repair, but the EPCs and EOC play a specific role in the formation of the blood vessels and the repair of the damaged tissue, and which signal pathways and regulatory mechanisms are still unclear. The Notch signaling pathway plays an important role in a number of systems from the development of the embryo to the adult, and plays a decisive role in the fate of the cells in different tissues The previous study suggests that the Notch signaling pathway plays an important role in the field planting, migration and homing of the EPC, and the earlier experiments have shown that the EPC can be migrated to the damaged liver tissue to participate in the regeneration of the liver tissue after the partial hepatectomy of the mouse, but the effect on the EOC is not In order to further analyze the regulation mechanism of Notch signaling pathway on endothelial stem/ progenitor cells, we study the regulation of Notch signaling pathway on the EPC of different stages of differentiation, because the transcription factor, RBP-J, is the key effect downstream of the Notch receptor. The transcription activation of the four Notch receptors in the nucleus can be mediated by the molecule, so it is regulated by the whole Notch signaling pathway. The results show that the Notch signaling pathway plays an important role in the maintenance of vascular balance and tissue repair by establishing a RBP-J conditional knockout mouse. main research The results were as follows:1. The number of EEPCs expressing CD34 +/ CD133 +/ VEGFR2 + from the bone marrow cells of mice was increased from the original 0.08% by the in vitro adherent amplification culture. up to 50% or more;2. We found that in In the three-dimensional lumen formation experiment, the EPCs can not form a tube-cavity-like structure, and the EOC can form a tube-cavity-like structure and is controlled by the Notch-RBP-J signal path; after blocking the Notch-RBP-J signal path, the proliferation and migration of the EEPC, the expression of the CXCR4 are all down, and the proliferation, the migration and the CXCR of the EOC The expression of the 4 is on the rise. The Notch signal path is prompted for the EEPC and The control of EOC was not the same;3. The mouse model was removed by the RBP-J conditional gene. We further observed that after the partial hepatectomy (PHx), the EEPC through the Notch signaling pathway could be quickly raised to the damaged liver. It was found that the regulation of the Notch-RBP-J signaling pathway in the liver regeneration process involved in the participation of EEPC and EOC was different, and EEPC and EOC were found to be in the liver regeneration. In conclusion, we removed the mice by RBP-J and observed the Notch-RBP-J signal for EEPC and EOC The results showed that the Notch signal was involved in the control of the regeneration of the liver cells, the repair of the liver, and the recovery of the liver function, and the proliferation, differentiation and migration of the EEPC and the EOC were regulated by the Notch-RBP-J signaling pathway, and the EEPC and EOC were in the liver. Different roles have been played in the regeneration process. These studies provide a further understanding of the function of endothelial stem/ progenitor cells, and in-depth analysis of Notch's effect on the endothelium/ progenitor cells
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

【共引文献】